Biochemical evidence supporting the presence of the classical mevalonate pathway in the thermoacidophilic archaeon Sulfolobus solfataricus

Abstract

The existence of the classical mevalonate (MVA) pathway was examined in the thermoacidophilic archaeon Sulfolobus solfataricus. The pathway is considered uncommon among archaea because the genes of the orthologues of phosphomevalonate kinase (PMK) and/or diphosphomevalonate decarboxylase (DMD) are absent in the genomes of most archaea. Instead, the modified MVA pathway, which involves isopentenyl phosphate kinase (IPK), has been proposed to exist in the archaea that lack the classical pathway. However, some archaea including S. solfataricus possess the genes of the orthologues of both IPK and all enzymes of the classical pathway. Biochemical characterization using recombinant proteins showed that the orthologues of the enzymes catalyzing the late steps of the classical pathway, i.e. MVA kinase, PMK and DMD, are all active. Moreover, in vitro conversion of the intermediates in the classical and modified pathways by cell-free extract from S. solfataricus indicated that only the classical pathway likely works in the organism.

In Vivo Formation of the Protein Disulfide Bond That Enhances the Thermostability of Diphosphomevalonate Decarboxylase, an Intracellular Enzyme from the Hyperthermophilic Archaeon Sulfolobus solfataricus

“…Soon after our discovery of the first archaeal DMD from S. solfataricus (22), we began an X-ray crystallographic study to reveal the structural differences between the enzyme derived from the hyperthermophilic archaeon and known DMDs from mesophilic bacteria and eukaryotes. Two different conditions for crystallization gave crystals with different space groups, having qualities that were sufficient for X-ray diffraction analysis (crystal DMD-P and DMD-AS) (Table 1).…”

“…Enzyme expression using E. coli Rosetta(DE3) transformed with the pET16b vector into which the sso2989 gene had been inserted (pET-SsoDMD) and partial purification with affinity chromatography using a 1-ml Histrap FF crude column (GE Healthcare) were performed as described in our previous study (22). The purified enzyme fraction containing DMD fused with an N-terminal polyhistidine tag was diluted with a 10-fold volume of a factor Xa cleavage buffer containing 50 mM Tris-HCl (pH 8.0), 0.1 M NaCl, and 5 mM CaCl 2 , which was then reacted with 400 U of factor Xa (Merck Millipore, Germany) at 22°C for 16 h. The solution was then loaded onto a Histrap FF crude column to remove the cleaved polyhistidine tag.…”

“…In the present study, we solved the crystal structures of diphosphomevalonate decarboxylase (DMD) from S. solfataricus, which had been discovered recently by us as the first archaeal DMD (22). DMD is an enzyme of the classical mevalonate (MVA) pathway and catalyzes the conversion of mevalonate diphosphate (MVAPP) into isopentenyl diphosphate (23,24), accompanying the hydrolysis of ATP to ADP and inorganic phosphate.…”

“…However, DMD is very rare in archaea, because almost all archaea utilize the modified MVA pathways that do not involve DMD (25,26). S. solfataricus is the only example of archaea that has been proven to date to possess the classical MVA pathway (22). The crystal structures of DMD have been solved with the enzymes from several eukaryotes (Saccharomyces cerevisiae [27], Trypanosoma brucei [28], Homo sapiens [29], and Mus musculus), and bacteria (Staphylococcus aureus [28], Staphylococcus epidermidis [30,31], Streptococcus pyogenes, and Legionella pneumophila).…”

Identification in Haloferax volcanii of Phosphomevalonate Decarboxylase and Isopentenyl Phosphate Kinase as Catalysts of the Terminal Enzyme Reactions in an Archaeal Alternate Mevalonate Pathway

“…Radiolabeling experiments suggest that the mevalonate (MVA) pathway provides the metabolites used in biosynthesis of archaeal membrane lipid (3). While a functional demonstration of the last two reactions of the classical mevalonate pathway has been shown in Sulfolobus solfataricus (4), other investigations have yet to identify enzymes catalyzing such reactions in most other archaea. This has prompted hypotheses that archaeal isoprenoids are synthesized, not by the classical MVA pathway (4) (Fig.…”